Rosmarinus officinalis L. hexane extract: phytochemical analysis, nanoencapsulation, and in silico, in vitro, and in vivo anti-photoaging potential evaluation

A shift towards natural anti-aging ingredients has spurred the research to valorize traditionally used plants. In this context, Rosmarinus officinalis L. was evaluated for its photoprotective, antioxidant, anti-inflammatory, and anti-wrinkling properties. GC/MS and LC-ESI-HRMS based phytochemical profiling of rosemary leaves hexane extract resulted in the identification of 47 and 31 compounds, respectively and revealed rich content in triterpenoids, monoterpenoids and phenolic diterpenes. In vitro assays confirmed the antioxidant, anti-aging, and wound healing potential of rosemary extract along with a good safety profile, encouraging further development. A systematic molecular modelling study was conducted to elucidate the mechanistic background of rosemary anti-aging properties through the inhibitory effects of its major constituents against key anti-aging targets viz. elastase, collagenase, and hyaluronidase. Development of rosemary extract lipid nanocapsules-based mucoadhesive gels was performed to improve skin contact, permeation, and bioavailability prior to in vivo testing. The developed formulae demonstrated small particle size (56.55–66.13 nm), homogenous distribution (PDI of 0.207–0.249), and negatively charged Zeta potential (− 13.4 to − 15.6). In UVB-irradiated rat model, topical rosemary hexane extract-loaded lipid nanocapsules-based gel provided photoprotection, restored the antioxidant biochemical state, improved epidermal and dermal histological features, and decreased the level of inflammatory and wrinkling markers. The use of rosemary hexane extract in anti-aging and photoprotective cosmeceuticals represents a safe, efficient, and cost-effective approach.


Results and discussion
Chemical profiling of R. officinalis hexane extract. GC-MS analysis. GC-MS analysis was implemented to assess the metabolite composition of rosemary hexane extract (RHE) revealing the presence of monoterpenes, their oxygenated derivatives, sesquiterpenes, long chain alkanes, and triterpenoids and resulting in the identification of 47 components representing 99.29% of detected peaks (Table 1). Triterpenes (45.2%) and hydrocarbons (41.8%) dominated the extract with α-amyrin, dotriacontane, β-amyrin, and triacontane as major components. Volatile constituents of rosemary aroma were mainly represented by monoterpenes and their oxygenated derivatives detected at 3% and 8.4%, respectively. The chief monoterpene was α-pinene, while the major oxygenated monoterpenes included the ketones verbenone and camphor, besides 1,8-cineole and borneol. This is congruent with previous reports of major terpenes of rosemary essential oil 11 . Lipophilic hexane extracts of aromatic plants exhibit aroma profile that closely mimics the characteristic odour of fresh plant. On the contrary, the aroma quality of volatiles prepared by distillation may differ from the fresh raw material because of the high temperatures used in distillation procedures 36,37 .
LC-ESI-HRMS analysis. Rosmarinus officinalis leaves hexane extract was profiled for its phytochemical composition using LC-ESI-HRMS. The compounds were tentatively identified by comparing their corresponding retention times and HRMS data with those previously reported in literature and online databases. The LC-ESI-HRMS analysis resulted in the tentative identification of 31 metabolites from different classes. Phenolic diterpenes (e.g., rosmanol, carnosol, carnosic acid, and rosmadial) represent the most abundant class detected in RHE with 14 identified compounds in accordance with previous studies 46, 47 , followed by triterpenoids (e.g., betulinic, 10 13 www.nature.com/scientificreports/ oleanolic and ursolic acids) represented here by 9 compounds. The identified compounds and their chromatographic and HRMS data characteristics are depicted in Table 2. Compounds 3, 4, 5, and 20 exhibited quasimolecular ions [M-H] − at m/z 345. Compounds 3, 4, and 5 were tentatively identified as rosmanol, (epi)(iso)rosmanol I, and (epi)(iso)rosmanol II based on the accurate masses of the observed deprotonated quasimolecular ions (m/z 345.171) and deprotonated dimers [2 M-H] − (m/z 691.3501). Additionally, fragment ions were detected at m/z 301, and 283 which correspond to the loss of CO 2 and sequential loss of CO 2 and water, respectively, in line with previously reported data for these isomeric compounds 46,48 . Compound 20 was identified as 12-O-methylcarnosic acid. Although it showed the same nominal mass with [M-H] − at m/z 345, it presented a different accurate mass of 345.2080 and eluted later in the chromatographic separation which is previously reported for 12-O-methylcarnosic acid 48,49 . Fragment ion at m/z 301 indicated the loss of CO 2 from the carboxylic acid group 48 .
Rosmadial (compound 8) and its isomeric form (compound 11) showed quasimolecular ions at m/z 343.1563 and 343.1565, respectively. A unique fragmentation pattern was recorded for these compounds; loss of ethylene, CO 2 and HCHO can account for the product ions observed at m/z 315, 299 and 313, respectively, as formerly described 46,48,49 . Compound 9 was identified as (epi)rosmanol methyl ether based on its [M-H] − accurate mass  46,51 .
Carnosic acid (compound 14) and its isomer (compound 21) were identified by their corresponding deprotonated quasimolecular ions observed at m/z 331.1928 and 331.1916, respectively. Carnosic acid produced a fragment ion at m/z 244 which can be attributed to the loss of CO 2 + CH 3 CH 2 CH 2 , as previously reported for this highly antioxidant phenolic diterpene 48,49 . In vitro antioxidant capacity. Oxidative stress is a key element in the aging process as well as in the aetiology of various chronic disorders through its inflammatory and degenerative consequences. Topical application of antioxidants can restore the balance between antioxidation and oxidation processes, prevent molecular damage, and maintain skin homeostasis. In this regard, plant extracts provide a wide range of antioxidant molecules ranging from phenolic acids, flavonoids, and tannins to carotenoids, tocopherols, and terpenoids. It is noteworthy that the extraction solvent has a strong impact on the antioxidant activity of a plant extract. Although polar solvents, e.g., methanol and ethanol, are best suited for extraction of polyphenols and better reflect the antioxidant potential of a plant 61 , the n-hexane extract of certain plants, including rosemary, demonstrated better antioxidant activity than their polar extracts based on in vitro chemical antioxidants tests 23,24,62 .
The antioxidant activity was assessed using DPPH and ABTS free radicals scavenging assays as well as FRAP assay. RHE was able to scavenge DPPH radical (IC 50 of 221.6 ± 11.8 µg/mL), but it was less active than Trolox (IC 50 of 6.1 ± 0.2 µg/mL) ( Table 3). The DPPH scavenging activity of RHE is comparable to Ipomoea cairica and Bauhinia purpurea hexane extracts 63 . However, it is much less than the DPPH scavenging capacity of rosemary essential oil (IC 50 of 77.6 µL/mL) 64 . Meanwhile, it is worth mentioning that this antioxidant activity is comparable and even superior to published antioxidant properties of phenolics rich extracts 65,66 .
The free radical scavenging capacity of RHE was also evaluated using the ABTS decolorization assay. This is based on the capacity of antioxidant compounds to scavenge the radical cation ABTS •+ relative to the standard antioxidant Trolox. RHE demonstrated a Trolox equivalent antioxidant capacity of 310.5 ± 12.3 mM TE/g dry extract (Table 3), well above the previously reported antioxidant capacity of Vitex agnus-castus leaves and fruits hexane extract 67 .
The FRAP assay is used to evaluate the reducing power of compounds and depends on the ability of antioxidants in plant extracts to reduce the colourless Fe 3+ -TPTZ complex to the blue coloured Fe 2+ -TPTZ complex. In FRAP assay, RHE demonstrated a Trolox equivalent antioxidant capacity of 394.7 ± 17.3 mM TE/g dry extract ( Table 3). The different antioxidant results obtained from the three assays may reflect differences in the capacity of compounds in the extract to quench DPPH and ABTS free radicals and to reduce ferric ion in vitro. Among the methods used, ABTS and FRAP assays are the most correlated, a finding that was formerly reported 68,69 . It should be noted that the antioxidant assays are preferably performed in the context of the whole organism to obtain more reliable information 70 , which is complemented in the present work by in vivo biochemical analysis.
In vitro anti-aging potential. Collagen, elastin, and hyaluronic acid represent the major structural components of the dermal extracellular matrix (ECM). Making up 80% of the skin dry weight, collagen is responsible for the skin tensile strength 71 . Brittle when dry but flexible and elastic when moist, elastin fibres maintain skin elasticity 72 . In addition, the mucopolysaccharide hyaluronic acid supports skin viscoelasticity, smoothness, and hydration 73 . Extrinsic skin aging is mainly attributed to the repeated exposure to solar UV radiation (photoaging), which causes overproduction of reactive oxygen species (ROS), leading to physical changes in the ECM. ROS are known to induce the expression of proteolytic enzymes, such as matrix metalloproteinases (MMPs), e.g., collagenase; serine proteases, e.g., elastase; as well as the glycosidase enzyme hyaluronidase [74][75][76] . These are key enzymes in the skin aging process, responsible for the degradation of collagen, elastin, and hyaluronic acid leading to remodelling of ECM and loss of skin elasticity.
To furnish preliminary insights regarding its anti-aging potential, rosemary extract was assessed for its in vitro anti-elastase, anti-collagenase, and anti-hyaluronidase activities. RHE demonstrated good dose-dependent inhibition of elastase activity with IC 50 value of 57.6 µg/mL close to the reference standard 1,10-phenanthroline (IC 50 = 25.6 µg/mL). The extract showed mild anti-collagenase and anti-hyaluronidase effects (IC 50 of 520.2 µg/ mL and 448.1 µg/mL, respectively) ( Table 4).
Interestingly, elastase enzyme has been reported to activate MMP precursors leading to further degradation of ECM 77 . Additionally, elastase was found to degrade decorin, a proteoglycan that binds to and protects collagen fibrils from cleavage by MMP. This renders collagen more susceptible to the proteolytic action of MMP 35 . Inhibition of elastase can hence stop subsequent degradation steps. Pentacyclic triterpenoids, e.g. lupeol and ursolic acid, are known to inhibit elastase 78 . Since collagenase is a zinc-containing enzyme, phenolic compounds, www.nature.com/scientificreports/ e.g. flavonoids, phenolic acids, phenolic diterpenes, tannins, and tocopherols (known as metal chelators), were reported to inhibit this metalloproteinase 79 .
In vitro wound healing potential. Wound healing includes the formation and remodelling of new tissues. Migration and proliferation of cells at the wound edge are necessary to close the wound and repair the injured tissue. Many plants have been used in folk medicine to accelerate this process and to prevent infection, such as calendula, for which the wound healing potential was established clinically 80 . In a wound-healing scratch assay, RHE improved the migration and repopulation of keratinocytes at the scratched area and considerably narrowed the scratched gap relative to the control. At  In silico molecular docking study. To rationalize elastase inhibition on a structural level, in silico docking study of the nine major constituents of RHE, as indicated by GCMS analysis, was performed revealing that two components, verbenone and α-amyrin, were able to bind effectively to elastase active site better than the control 1,10-phenanthroline with a Glide G-score of −5.327 and −4.563 kcal/mol, respectively, compared to −4.556 kcal/mol for 1,10-phenanthroline ( Table 5). The superior score, especially for verbenone, explains the good inhibitory effect of RHE against elastase enzyme in vitro. Indeed, verbenone was previously reported as potent inhibitor of elastase enzyme with IC 50 in the picomolar range 86 . The remaining 7 constituents showed docking scores lower than the control and were therefore excluded as being solely responsible for the in vitro anti-elastase activity of RHE. The docking pose of verbenone in elastase revealed the formation of a major hydrogen bond with the backbone amide nitrogen of Val224 through its carbonyl group (Fig. 1A,B). This critical interaction plays a major role in the anchorage of verbenone to the binding site of the enzyme. The rest of the small hydrophobic skeleton then buries itself in the binding site groove forming Van der Waal interactions with the hydrophobic side chains of surrounding residues, with minimal exposure to the aqueous medium. Due to verbenone small size and the presence of a hydrogen bond donor, it can fit easily in elastase active site and form non-covalent interactions that promote enzyme inhibition.
In contrast, the docking poses of verbenone in collagenase and hyaluronidase binding sites are unfavourable. In collagenase, verbenone formed two hydrogen bonds with the backbone nitrogen of Leu185 and Ala186, along with Van der Waal interactions with nearby hydrophobic residues, e.g., Leu185 and Tyr244. However, verbenone failed to interact with the active site zinc, which is crucial for collagenase inhibition. Due to this lack of ligand-metal coordination, verbenone failed to inhibit collagenase enzyme (Fig. 1C,D).
In hyaluronidase binding site, verbenone formed a hydrogen bond to the side chain hydroxyl group of Ser303 (Fig. 1E,F which constitutes an important residue in the binding of hyaluronic acid to hyaluronidase as shown in the co-crystal structure (PDB ID: 1FCV). Furthermore, per-residue interaction scores showed that the hydrogen bond strength of verbenone to Ser303 in hyaluronidase (−0.320 kcal/mol) is greater than hydrogen bond strength of verbenone to Val224 in elastase (−0.286 kcal/mol). However, the overall binding pose of verbenone to hyaluronidase does not qualify it to become an inhibitor due to the superficial binding of verbenone to the active site of hyaluronidase, prohibiting the ligand from immersing itself in the folds of the active site and escaping the aqueous medium. The over-exposure of the hydrophobic skeleton of verbenone to the aqueous medium has made the overall binding of the ligand unstable, and therefore its activity minimal.
It is noteworthy that in collagenase and hyaluronidase docking experiments, the control 1,10-phenanthroline scored higher than RHE major constituents (Table 5), among which camphor showed the best score and binding pose. The docking experiment of camphor in collagenase binding site showed better binding compared to verbenone. Camphor directed its carbonyl oxygen towards the active site zinc and approached it at 1.98 Å. At this distance, the carbonyl could form a mono-dentate metal chelation interaction with the active site zinc ( Fig. 2A,B). Furthermore, the hydrophobic skeleton of camphor was buried among the hydrophobic residues Pro242, Ile243, Leu185, Tyr244, and Tyr214. Despite the good binding pose of camphor in the binding site of collagenase, the www.nature.com/scientificreports/ expected activity of camphor against collagenase may not be ideal due to the lack of another metal chelation interaction with zinc, since a bi-dentate metal coordination is required for potent collagenase inhibition.
In case of hyaluronidase, camphor formed of hydrogen bonds. The carbonyl oxygen of camphor bonded to the hydroxyl side chain of Ser303 and the backbone amide nitrogen of Ser304 (Fig. 2C,D). Nevertheless, camphor binding to hyaluronidase could not be stable due to the exposure of the hydrophobic skeleton of camphor to the aqueous medium. These findings are in agreement with the in vitro anti-aging results. Verbenone, with its higher docking score and binding mode, may be responsible for the overall anti-elastase activity of the extract. Table 5. Docking scores of RHE major constituents against elastase, hyaluronidase and collagenase as compared to the control, 1,10-phenanthroline. *Compounds were rejected by the docking engine due to their exceedingly large size. Characterization of LNC. The results of the PS distribution and ZP measurements are listed in Table 6.
As shown, the PS of blank LNC, 4% RM-LNC (rosemary-loaded lipid nanocapsules), and 10% RM-LNC were 42.28 ± 0.417 nm, 55.20 ± 0.218 nm, and 64.81 ± 1.113 nm, respectively. The obtained PS of blank LNC was comparable to previous results regarding the small size of LNC 87,88 . As observed, rosemary loading into LNC resulted in a significant (p < 0.05) increase in PS of RM-LNC, which may be attributed to the increase in the mass of the oily core with subsequent increase in PS 89 . Both blank and rosemary-loaded LNC showed homogenous PS distribution as reflected by the low PDI values (< 0.3). Despite rosemary loading into LNC resulted in significant (p < 0.05) increase in PDI values, they still reflect the narrow particle distribution.  www.nature.com/scientificreports/ The small PS associated with homogenous distribution is a main characteristic of LNC, which favours its application in topical use. Small PS represents a main advantage due to the increased surface area of the particles forming a dense monolayer and promoting better skin contact, which is needed to achieve high protection against UV radiation 90 . Moreover, previous studies reported the enhanced penetration of the lipid nanocarriers into the epidermal layer as the PS decreases 87,91,92 .
The ZP values of blank LNC, 4% RM-LNC, and 10% RM-LNC were −12.6 ± 1.41 mV, −13.1 ± 0.63 mV, and −15.4 ± 2.62 mV, respectively. The negative charge of the prepared LNC is attributed to the existence of negatively charged phospholipids and the PEG dipoles that took part in the formation of the LNC shell [93][94][95] . It has been reported that colloidal stability increases as the ZP values increase (≥ ± 30 mV), which is attributed to the electric repulsion between particles. However, the stability of LNC with low ZP values is attributed to steric stabilization of LNC by their tensioactive rigid membrane 33,34 . It can be observed that rosemary-loaded LNC displayed higher negative value of the ZP than blank LNC. A clear explanation was provided by a previous study conducted by Valcourt et al. 2016, which stated that the incorporation of oils into the lipid core of the LNC had a negative impact on the density of PEGylated surfactant at the particle surface with a subsequent increase in the contact area between the lipoid molecules and the external phase and resulted in an increase in the absolute value of the ZP 89 .  www.nature.com/scientificreports/ Characteristics of RM-LNC gels. Incorporation of lipid nanocarriers into mucoadhesive gels combined the advantages of a topically delivered formulation with those of nanocarriers in the same final product. These advantages include ease of application, high mucoadhesion with extended skin contact, and slow drug release rate 96,97 . Based on our previous study, 3% w/w HEC had a viscosity of 30 to 40 poise 98 , which is considered acceptable viscosity for sunscreen gels as reported in previous study 99 . Therefore, 3%w/w HEC was chosen to be added to the LNC dispersion. It was expected that the presence of LNC might have an influence on the measured viscosity of the gel; therefore, the rheological properties of the RM-LNC gels were investigated. In addition, RM-LNC gel was characterized in terms of PS, PDI, and ZP.
Blank LNC, 4%RM-LNC, and 10%RM-LNC gels showed PS equals 45.72 ± 0.079 nm, 56.55 ± 0.384, nm and 66.13 ± 1.306 nm, respectively, where no significant (p > 0.05) increase in PS was reported upon LNC incorporation into HEC gel. As observed, all LNC gels are characterized by low PDI values and negatively charged ZP, which indicates their physical stability upon addition of the gelling agent.
The respective viscosity of blank LNC, 4%RM-LNC, and 10%RM-LNC gels were 32.91 ± 1.54, 35.42 ± 3.89, and39.55 ± 2.76 poise, respectively, which are acceptable values for the topical application. The pH values of blank LNC, 4%RM-LNC, and 10%RM-LNC gels were in the range of 6-8, which are considered safe for application as a sunscreen for its photoprotective effect.
In vivo studies. Biochemical analysis. Unprotected exposure to UVB irradiation results in skin damage; this can be assessed by the validation of different biochemical markers. In this study, the suggested protective effect of the prepared RM-LNC in comparison to RHE was investigated by measuring the level of some antioxidant, anti-inflammatory, and anti-wrinkling markers.
Antioxidant markers. The non-enzymatic antioxidants, GSH, as well as the antioxidant enzymes, SOD and CAT, were measured in the different studied groups. It is expected that the level of these enzymes decreases in oxidative stress conditions, like exposure to UV irradiation. As listed in Table 7, the marked decrease in their level in case of the positive control group compared to the negative one (p < 0.05) was abolished in the groups administered with the RHE as well as the RM-LNC gels, suggesting that rosemary can replenish antioxidants. This validates the initial in vitro screening, sheds light on skin penetration of applied formulae, and confirms their effectiveness in biological systems. The more potent effect of RM-LNC gels compared to the RHE (p < 0.05) is expected based on the above discussed in vitro study as well as the visual examination of the dorsal rats' skin before sacrificing. The enhanced RHE solubilization and release, reduced PS, as well as the elastic properties of the designed LNC can assure an enhanced skin penetration and photoprotective effect. Some previous studies have reported that the PS range recorded for prepared formulae facilitates drug penetration and accumulation into the skin, which allows a localized and site-specific drug effect 87,100 .
Anti-inflammatory activity. UVB exposure up-regulates inflammatory cytokines causing skin damage 101 . The values seen in Table 7 show that UVB exposure induced a significant increase in the inflammatory markers (IL-1β, IL-6, and NF-kB) of the positive control group compared to those of the negative control (p < 0.05). This effect decreased in case of the groups pre-treated with RHE and RM-LNC (p < 0.05). However, the effect of the RM-LNC formulae was higher than the RHE (p < 0.05). As discussed above, the superior effect of the RM-LNC against the inflammatory reactions induced by UVB irradiation can be due to the intrinsic properties of the designed LNC, which can breach the skin barrier and penetrate deeply into the inner skin layers. This reduction in photo-inflammation was evidenced by decreased erythema, edema, and skin thickness. The observed antiinflammatory effect can be attributed to the rich triterpenoids and phenolic diterpenes content of rosemary. Table 7. Effect of UVB-irradiation and different formulations on the oxidative stress, inflammatory and wrinkling markers in rats. NC: Negative control (normal rats), PC: positive control (subjected to UVB irradiation and received no treatment), while T1, T2, T3 and T4 received RHE, 4%-RM-LNC gel, 10% RM-LNC gel and plain LNC gel. Each value is presented as mean ± standard error of the mean (SE) for 10 rats. *Statistically significantly different from the normal control group (P < 0.05). @ Statistically significantly different from the positive control group (P < 0.05). # Statistically significantly different from the T1 group (P < 0.05). a Statistically significantly different from the T2 group (P < 0.05). +++ Statistically significantly different from the T3 group (P < 0.05).
Anti-wrinkling markers. Exposure to UVB irradiation triggers the production of free radicals, which upregulate the production of matrix metalloproteinases (MMPs). Degradation of the collagen and elastin network is caused by MMPs 106 , leading to skin wrinkling. UVB irradiation causes keratinocytes to secrete IL-1, which stimulates GM-CSF secretion and triggers fibroblasts to stimulate their expression of neprilysin/NEP, which results in the deterioration of the three-dimensional fibre networks and the loss of skin elasticity and wrinkles formation 107,108 . As observed in Table 7, the levels of MMP1, GM-CSF, neprilysin, and elastase are higher in the positive control group than in the negative control one (p < 0.05). Application of RHE or RM-LNC protected the skin from wrinkling and aging (p < 0.05). As expected, the photoprotective effect of the studied formulae was significantly superior (p < 0.05). Surprisingly, the major ingredient in RHE, α-amyrin, did not provide in vitro protection against UVB damage in earlier reports 43 , suggesting that the observed photoprotection can be mediated by potentiating interactions among several constituents, including the minor ones.
Cutaneous irritancy test. Skin applied formulae are required to be innocuous, neither creating irritancy nor allergenicity 109 . Both the plain and rosemary-loaded LNC gels were tested to evaluate the safety of each component of the preparations. The results shown in Table 8 proved the non-irritancy of tested gels (PII < 2) all over the period of the experiment (72 h). Statistical analysis shows that the formalin solution (group 2) was significantly irritant (p < 0.001) compared to the control group (group 1) and all the gels, whereas there was no significant difference (p > 0.05) between all gels and the control group.
Histopathological examination. The photos of the skin subjected to histopathological study are displayed in Fig. 3. The negative control group demonstrated normal morphological features of skin layers including thin intact epidermal layer with well-organized apparent intact subcellular details of different keratinocytes in different zones, intact dermal layer with abundant collagen fibres, minimal inflammatory cells infiltrates, and normal vasculatures. On the other hand, the positive control group revealed significant increase of epidermal thickness with alternated areas of apparent intact or pyknotic basal cells layer accompanied with mild to moderate dermal mononuclear inflammatory cells infiltrates as well as congested subepidermal blood vessels and focal hemorrhagic zones. A certain improvement of the condition can be observed in rats treated with RHE; however, samples revealed moderate reduction of epidermal thickening with persistence of degenerative changes records of basal cell layer, mild subepidermal mononuclear cells infiltrates, and congested blood vessels. Group T2 samples showed moderate reduction of epidermal thickening as shown in Fig. 3, with persistence of degenerative changes records of basal cell layer and mild subepidermal mononuclear cells infiltrates. However, normal subepidermal vasculatures were recorded.
Group T3 samples demonstrated almost intact well-organized skin layers with minimal records of abnormal morphological features all over epidermal and dermal layers.
Finally, group T4 showed abundant records of degenerated and pyknotic epidermal keratinocytes with normal epidermal thickness ranges accompanied with mild occasional sub-epidermal inflammatory cells infiltrates as well as congested BVs.
Previous studies have established the anti-inflammatory and photoprotective activities of rosemary polyphenols and polar extracts 19,110 . However, this is the first report of the photoprotective potential of rosemary hexane extract which is often regarded as agro-industrial processing waste during the extraction of polyphenols. www.nature.com/scientificreports/  www.nature.com/scientificreports/ 130 V, respectively, and collision energy was 10 V. The nebulization pressure was 58 psi. Data processing was performed using MassHunter workstation B.06.00 (Agilent Technologies, 2012) and compounds were tentatively identified according to their mass spectra, accurate mass and retention time, in comparison with literature.
In vitro antioxidant activity. DPPH radical scavenging activity. The scavenging activity of the stable 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical by RHE was assessed according to the method reported by Boly et al. 112 .
ABTS radical scavenging assay. This assay was carried out according to the method previously reported by Arnao et al. 113 .
FRAP assay. The method is based on the reduction of a ferric-tripyridyltriazine (Fe 3+ -TPTZ) complex to its intensely blue ferrous form, at low pH, as previously described 114 .
In vitro anti-aging potential. Anti-elastase assay. The elastase inhibitory activity was assessed fluorimetrically using the EnzCheck® elastase assay kit (Molecular Probes, Laiden, Netherlands). A 1 mg/mL stock solution of the substrate (DQ elastin) was prepared in deionized water. Porcine pancreatic elastase stock solution was prepared in deionized water at 100 U/mL and dilutions were made in Tris-HCL buffer. Pre-incubation of 50 µL of different dilutions of the extract (inhibitor) with 100 µL of the enzyme was done for 15 min followed by the addition of the substrate (50 µL). Controls were prepared with buffer instead of extract. Fluorescence intensity was continuously measured for 20 min in a fluorescence microplate reader equipped with standard fluorescein filters (λ ex 505 nm, λ em 515 nm). Subtraction of background fluorescence was done in no-enzyme wells. 1,10-Phenathrolinewas used as a standard elastase inhibitor. The IC 50 is the concentration of the extract required to inhibit 50% of elastase activity.
where S is the corrected fluorescence of the extract samples and C is the corrected fluorescence of the control.
Anti-collagenase assay. The evaluation of collagenase inhibitory activity was performed fluorimetrically in a microplate reader. Briefly, the self-quenched BODIPY conjugate of gelatin (Type B) was used as a fluorogenic substrate to monitor the activity of collagenase (Biovision, CA, USA). Collagenase stock solution was prepared in 50 mM Tricine buffer at 0.8 U/mL and the substrate (BODIPY) was dissolved in Tricine buffer to 2 mM. One microliter of different concentrations of the extract (1, 10, 100, and 1000 µg/mL) was incubated with the 5 µL collagenase in buffer and 44 µL Tricine buffer for 15 min before adding the substrate. (1, 10)-Phenanthroline was used as a positive control. The reaction is initiated by mixing two µL of the substrate with the previous reaction mixture. Negative controls were prepared with the buffer. Fluorescence intensity was monitored (λ ex 490 nm, λ em 520 nm, 515 nm cut-off) in a kinetic mode at 37 °C for 30-60 min.
where S is the corrected fluorescence of the extract sample and C is the corrected fluorescence of the control.
Anti-hyaluronidase assay. A turbidimetric assay was performed to assess the hyaluronidase inhibitory activity using QuantiChrom™ Hyaluronidase Inhibitor Screening Assay Kit (BioAssay Systems, CA, USA). Bovine hyaluronidase (type-1-S, Sigma Aldrich, St. Louis, MO, USA) was diluted to 10 U/mL in buffer. From this solution, 40 µL were transferred to a 96-well plate. Instead, enzyme buffer (40 µL) was used for No Enzyme Control (NEC), while hyaluronidase (40 µL) was used for No Inhibitor Control (NIC). To the NIC and NEC wells, 20 μL DMSO were added and to the sample wells 20 µL of the desired extract concentrations were added followed by incubation for 15 min at room temperature. The substrate (40 µL) was added and the plate was incubated for 20 min. The stop reagent (160 µL) was used to halt the enzymatic reaction and forms turbidity with any residual hyaluronic acid. Plate was incubated for 10 min and the optical density was read at 600 nm. The percentage inhibition was calculated as follows: where OD NEC , OD NIC , and OD sample represent the optical density values of the No Enzyme Control, No Inhibitor Control, and the extract.
Scratch-wound healing assay. Effect of RHE on keratinocytes migration was evaluated using the scratch assay as previously described 115 .
In silico molecular modelling. All  www.nature.com/scientificreports/ 1FCV, 2.65 Å), were downloaded from the Protein Data Bank (PDB). The crystal structures were co-crystallized with non-covalent inhibitors. The PDB files were imported into Maestro and prepared using the protein preparation wizard and standard protein preparation protocol. The docking grid was then generated using the grid receptor grid generation module and the co-crystallized ligand was selected as the grid centre. The ligands were then imported and prepared using Ligprep module and standard ligand preparation protocol. Molecular docking was carried out using Glide Standard Precision and no constraints. Per-residue interaction scores were calculated for selected ligands and residues during docking re-runs using the same procedures.
Preparation of blank and rosemary-loaded LNC. Blank LNC were prepared using the phase inversion method with three temperature cycles 87 . In brief, aqueous phase composed of Solutol® HS 15 (1 g), sodium chloride (0.1 g), and demineralized water (3 g) was mixed with the oily phase of Labrafac (0.9 g) and EP (0.1 g) in a closed container under magnetic stirring for 10 min. The mixture was heated up to 85 °C under magnetic stirring, followed by cooling to 55 °C to ensure phase inversion from w/o emulsion to o/w emulsion. The heating/cooling cycle was repeated two times followed by the addition of 5 ml of cold water (0-2 °C) with magnetic stirring. The LNC dispersions obtained were kept at 4 °C for further investigation. Rosemary-loaded LNC (RM-LNC) were prepared using the same procedure, where the rosemary extract (4%w/w or 10%w/w) was dissolved in the oily phase by magnetic stirring before mixing with the aqueous phase, then the procedure was completed as previous.
Particle size distribution and zeta potential measurements of the prepared LNC. The particle size (PS), polydispersity index (PDI), and zeta potential (ZP) for the prepared blank and RM-LNC were determined at 25 °C using a laser diffraction particle size detector (Zetasizer; Malvern Instruments, Malvern, UK) after suitable dilution.
Preparation and characterization of blank and RM-LNC-based gels. Gels based on LNC were prepared by gradual sprinkling of HEC as gelling agent into the LNC dispersions under magnetic stirring until complete hydration. The gel was sonicated for dissipation of entrapped air and stored at 4 °C for further evaluation 117 . The PS, PDI, and ZP of blank and RM-LNC-based gels were measured as previously described after suitable dilution with deionized water with magnetic stirring. Viscosity measurements were conducted using viscometer (Brookfield Engineering Laboratories Inc., Model HADV-II, USA) connected to a digital thermostatically controlled circulating water bath (Polyscience, Model 9101, USA) with spindle 52 at a speed of 50 rpm 25 ± 0.1 °C. Equilibration of the sample for 5 min was made following loading of the viscometer. All studies were performed in triplicates and the average was taken 118 . The pH of 5% w/w dispersions of the gels in water was determined using pH meter.
In vivo study. Animals. The experiment was performed on the hairless skin of adult male Wistar rats weighing between 180 and 220 g (6-8 weeks old) obtained from the animal house of the National Research Center, Cairo, Egypt. The animals were housed in plastic cages and kept in a conditioned atmosphere at 22 ± 3 °C and humidity 50-55% with 12 h light/dark cycles. They were fed standard pellet chow (El-Nasr chemical company, Cairo, Egypt) and were permitted free access to water. This study was conducted in accordance with ethical procedures and policies approved by the Institutional Animal Care and Use Committee of Cairo University, Egypt (Ethical Approval Number IACUC-CU-III-F- . The study followed the recommendations in the ARRIVE guidelines.
Experimental design. The dorsal side of the rats was shaved 24 h before the beginning of the experiment.
Sixty rats were randomly divided into six groups, each containing 10 animals: a negative control group (C1) was not exposed to irradiation; a positive control group was subjected daily to UVB irradiation for 10 consecutive days and received no treatment. The other four groups named T1, T2, T3, and T4 received RHE, 4%-RM-LNC gel, 10% RM-LNC gel, and plain LNC gel, respectively, daily one hour before the UVB exposure A UV lighter (peak emission was 302 nm, CL-1000 M, UVP, Upland, CA, USA) was used for UVB irradiation. UVB irradiation doses were 40-80 mJ/cm 2 (exposure time was 15-30 s) and the lamp was fixed 5 cm above the platform where rats were placed 119 . Tissue preparation. At the end of the experiment, rats were anesthetized by ketamine (85 mg/kg, i.p.), euthanized by cervical dislocation, and the treated skin of each rat was dissected out into two halves. The first half of the dorsal skin of rats was preserved in 10% formalin for histopathological examination. The other half of skin samples were homogenized and subjected to biochemical estimation of the antioxidant, anti-inflammatory, and anti-aging activities of the prepared RM-LNC gels in comparison with the RHE.
Biochemical analysis. Antioxidant activity. The level of catalase (CAT), reduced glutathione (GSH), and superoxide dismutase (SOD) reactive substances was estimated as reported previously 120,121 in the homogenate. Catalase (CAT) ELISA Kit was purchased from Hubei, China, Rat Superoxide Dismutase (SOD) ELISA Kit was purchased from MyBioSource, San Diego, US, and Glutathione peroxidase (GSH) was purchased from Shang-Hai BlueGene Biotech CO., China.
Histopathological study. Skin specimens obtained from the rats' dorsal skin were fixed and embedded in paraffin for histopathological studies. Paraffin bees wax tissue blocks were prepared for sectioning at a thickness of four µm by sledge microtome. The obtained tissue sections were collected on glass slides, de-paraffinized, and then stained by hematoxylin and eosin stain for examination using the light electric microscope (Optika B 150, Optika Microscopes, Italy).

Cutaneous irritation.
The irritancy of the RM-LNC gels was evaluated according to the method previously described 122 . The dorsal side of the rats was shaved 24 h before the beginning of the experiment. The animals were divided into 6 groups each containing 6 rats: Group 1 served as control (no treatment), group 2 received 0.8% v/v aqueous formalin solution as a standard irritant 123 , groups T1, T2, T3, and T4 received RHE, 4%-RM-LNC gel, 10% RM-LNC gel, and plain LNC gel. An amount of 100 mg gel (or formalin solution) was applied once daily for 72 h. The application sites were examined for edema and erythema at 24 and 72 h and graded (0-4), as shown in Table 9, according to a visual standard score 124 ; the final score represents the average of the 24 and 72 h readings. The primary irritancy index (PII) was determined for each preparation by adding the edema and erythema scores; the formulations were accordingly classified as non-irritant if PII < 2, irritant if PII = 2-5, and highly irritant if PII = 5-8.

Statistical analysis.
The invitro data were compared using one-way analysis of variance, followed by multiple comparisons of Tukey-Kramer test using Graph Pad Instat® software (GraphPad4 Software, La Jolla, CA). The significance level was at p < 0.05. Data obtained from in vivo study were expressed as the mean of three experiments ± the standard deviation (SD) or ± the standard error of mean (SEM) and were analysed using oneway analysis of variance (ANOVA), followed by the least significant difference procedure using SPSS® software (SPSS, Inc., Chicago,Illinois, USA). Statistical differences yielding p < 0.05 were considered significant.

Conclusions
Bioactive natural products and plant extracts inspired by traditional medicine are increasingly expanding the anti-aging and photoprotective therapeutic arsenal especially with the increasing life expectancy and the green shift towards the use of natural health care products. With its content of phenolic diterpenes, triterpenoids, monoterpenoids, and long chain hydrocarbons, rosemary hexane extract demonstrated interesting in vitro antielastase, antioxidant, and wound healing properties associated with no cytotoxicity, representing a cost-effective and relatively safe anti-aging approach. In silico molecular modelling posed verbenone as the main constituent responsible for the anti-elastase activity of the extract through its significantly high docking score and favourable binding mode. The findings were further consolidated with in vivo results where Rosmarinus officinalis hexane extract, formulated in lipid nanocapsules-based mucoadhesive gel, provided UV-protection, restored the antioxidant biochemical state, decreased the level of inflammatory and wrinkling markers, and improved epidermal and dermal histological features in UV-irradiated rat model. The feasibility of synergy with known antioxidant and photoprotective natural products and the use of systemic photoprotection in conjunction with topical routes are yet to be explored. Severe erythema and scar formation 4 Edema formation (Ed.) None 0 Very slight edema 1 Slight edema (edges of area well defined by definite raising) 2 Moderate edema (area raised approximately 1 mm.) 3 Severe edema (raised more than 1 mm. and extending beyond area of exposure 4